Dissolved organic phosphorus bond-class utilization by Synechococcus

Abstract Dissolved organic phosphorus (DOP) contains compounds with phosphoester, phosphoanhydride, and phosphorus–carbon bonds. While DOP holds significant nutritional value for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by Synechococcus strains from open and coastal oceans. Both strains exhibited comparable growth rates when provided phosphate, a phosphoanhydride [3-polyphosphate and 45-polyphosphate], or a DOP compound with both phosphoanhydride and phosphoester bonds (adenosine 5′-triphosphate). Growth rates on phosphoesters [glucose-6-phosphate, adenosine 5′-monophosphate, bis(4-methylumbelliferyl) phosphate] were variable, and neither strain grew on selected phosphorus–carbon compounds. Both strains hydrolyzed 3-polyphosphate, then adenosine 5′-triphosphate, and lastly adenosine 5′-monophosphate, exhibiting preferential enzymatic hydrolysis of phosphoanhydride bonds. The strains’ exoproteomes contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5′-triphosphate under phosphate deficiency, suggests active mineralization of phosphoanhydride bonds by these exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity toward the phosphoanhydride 3-polyphosphate, with varying affinities between strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition and highlight varied growth and enzymatic responses to molecular diversity within DOP bond-classes, thereby expanding our understanding of microbially mediated DOP cycling in marine ecosystems.


Introduction
The picoc y anobacterium Synechococcus is an abundant photosynthesizer, inhabiting multiple climate zones, as well as open ocean and coastal regions (Palenik et al. 2006, Zwirglmaier et al. 2008, Tai and Palenik 2009, Sohm et al. 2016, Bock et al. 2018, Nagarkar et al. 2021 ).Phosphorus (P) availability is a dominant factor influencing Synechococcus ecophysiology and abundance, as phosphate (P i ) can be present at biologically low, colimiting, and limiting concentrations in surface mixed-layer oligotrophic regions (Krom et al. 2010, Lomas et al. 2010, Kretz et al. 2015, Djaoudi et al. 2018, Sosa et al. 2019, Yuan et al. 2024 ).As one strategy to cope with P i scarcity, marine micr oor ganisms, including Synec hococcus , use dissolv ed or ganic phosphorus (DOP), whic h typicall y constitutes the dominant fraction of the dissolved P pool in open ocean surface waters (Lomas et al. 2010, Duhamel et al. 2014, 2021, Karl and Björkman 2015, Ranjit et al. 2024 ).Even in P i -replete regions, the labile fraction of DOP is rapidly recycled (Benitez-Nelson andBuesseler 1999 , Nausch et al. 2018 ), emphasizing the role of DOP in microbial P nutrition and its potentially significant role in sustaining primary productivity (Björkman et al. 2018, Whitney and Lomas 2019, Duhamel et al. 2021, Letscher et al. 2022 ).
Natural marine DOP can be classified into three P bond-classes: phosphoesters (P-esters), polyphosphates (PolyP), and phosphonates (Phn) (Kolowith et al. 2001 , Young andIngall 2010 ).P-esters ( + V oxidation state), typically in the form of monoesters (P-O-C) and diesters (C-O-P-O-C), are the most abundant ( ∼80%-85% of the high molecular weight dissolved organic matter) (Young and Ingall 2010 ).P-ester bioavailability has historically focused on monoesters (Moore et al. 2005, Wang et al. 2016, Filella et al. 2022 ), though there is increasing support that certain marine species can utilize diesters (Yama guc hi et al. 2005 , Hull andRuttenberg 2022 ).PolyP is a polymer composed of orthophosphate repeating units linked by phosphoanhydride (P-O-P) bonds and is estimated to account for ∼8%-13% of the high molecular weight dissolved organic matter (Young and Ingall 2010, Diaz et al. 2016, Saad et al. 2016 ).While PolyP can be found in organic and inorganic forms, it is typically measured in the organic P pools as it requires prior hydr ol ysis to yield soluble r eactiv e P (Armstr ong et al. 1966, Karl and Tien 1992, Karl and Björkman 2015 ).Although poorly characterized, Pol yP quantifications r e v ealed similar r elativ e concentr ations in the North Pacific Subtropical Gyre (Diaz et al. 2008(Diaz et al. , 2016 ) ) and Indian Ocean (Martin et al. 2018 ) surface waters.Phosphonates (P-C) include P in its + III oxidation state and account for ∼5%-10% of the high molecular weight dissolved organic matter (Young and Ingall 2010 ).Natur all y and artificiall y occurring phosphonates have the potential to be bioavailable P sources for certain marine bacteria (Repeta et al. 2016, Sosa et al. 2019 ), marine c y anobacteria (Ilikchy an et al. 2009(Ilikchy an et al. , 2010 ) ), and marine eukaryotic taxa (Wang et al. 2016 , Whitney andLomas 2019 ).Despite the importance of DOP, the r elativ e bioav ailability of specific bond-class compounds is poorly resolved (Karl and Björkman 2015, Diaz et al. 2016, Granzow et al. 2021 ).
To acquire P i from DOP, marine microorganisms can use Phydrolases, including alkaline phosphatases (AP), which are regulated by the Pho Regulon, itself controlled by P i availability (Cembella et al. 1984, Duhamel et al. 2010, 2014, Santos-Beneit 2015, Huang et al. 2018, Li et al. 2019, Sisma-Ventura and Rahav 2019 ).APs include isoforms (phoA, phoD, and phoX) that are widely distributed in prokaryotes, including Synechococcus (Tetu et al. 2009 , Cox andSaito 2013 ), and are known for hydrolyzing Pmonoesters, and possibly P-diesters (Huang et al. 2018, Sriv astav a et al. 2021 ).The enzymes responsible for Pol yP degr adation ar e not well-c har acterized, though ther e is incr easing e vidence that marine APs may be able to hydr ol yze Pol yP (Martin et al. 2018, Lin et al. 2019, Adams et al. 2022 ).Certain coastal Synechocococus str ains, namel y CC9311 and CC9902, lac k the Pho Regulon.This absence has been hypothesized to be an adaptation to their growth in P-replete environments (Su et al. 2007 ).The lack of this regulatory complex contrasts with its identification in both open ocean WH8102 and coastal WH5701 Synechococcus strains (Su et al. 2007, Scanlan et al. 2009, Tetu et al. 2009, Christie-Oleza et al. 2015, Santos-Beneit 2015 ).The Pho Regulon also controls the expression of the phn operon, a multigene complex that allows for the transport and use of phosphonates (Kamat and Raushel 2013, Tiwari et al. 2015, Stosiek et al. 2020 ).The phn operon encodes a range of P-C clea ving enzymes , including the P-C lyase complex, which supports the hydrolysis of a range of phosphonates.Additional enzymes, such as phosphonohydrolases, act on phosphonates independently of P i availability and are present in a range of prokaryotes (McGrath et al. 1997, Benitez-Nelson et al. 2004, Quinn et al. 2007, Villarreal-Chiu et al. 2012 ).
Though P-esters are thought to dominate microbial DOP nutrition, recent studies using culture isolates indicate that to some micr obial gr oups , P olyP pla ys an important role (Lin et al. 2016, Diaz et al. 2018, 2019, Duhamel et al. 2021, Adams et al. 2022 ).Specifically, picoc y anobacteria strains Prochlorococcus MED4, MIT9312, and MIT9313, and Synec hococcus WH8102 can gr ow on short-c hain 3-pol yphosphate (3-Pol yP) as a sole source of P (Moore et al. 2005 ).For marine bacteria cultures of Ruegeria pomeroyi DSS-3, Pol yP and P-ester substr ates can support equiv alent gr owth (Adams et al. 2022 ), while diatom cultures of the genus Thalassiosira exhibit pr efer ential degr adation of Pol yP ov er P-esters (Lin et al. 2016, Diaz et al. 2018, 2019 ).Considering the widespread pr esence of Synec hococcus and its importance in biogeochemical c ycling, w e assessed bond-specific bioavailability and utilization of DOP compounds to open ocean (WH8102) and coastal (WH5701) Synec hococcu s str ains.We hypothesized that Synec hococcus can hydr ol yze both P-anhydrides and P-esters, possibly using AP, supporting a flexible P metabolism favoring their wide distribution across global surface waters.

Synechococcus gr owth, ax enicity, and cell counts
Axenic Synechococcus WH8102 (open ocean strain) and WH5701 (coastal strain) were obtained from the National Center for Marine Algae and Microbiota (NCMA, Bigelow Laboratories, East Boothbay, Maine).Both strains were grown in batch culture using SN media (Waterbury et al. 1986 ) made with a ged, filter ed (0.2 μm), and autoclaved (120 • C, 30 min) seawater from station ALOHA (A Long-term Oligotrophic Habitat Assessment).At the late-exponential phase, cultur es wer e tr ansferr ed in triplicate to one of two SN media: (1) + P i (45 μmol l −1 KH 2 PO 4 ; following Waterbury et al. 1986 ) and (2) −P i (no KH 2 PO 4 added; P i below detection limit).All cultures were incubated at 25 • C on a 12 h:12 h light cycle at 130 μmol m −1 s −1 in sterile culture flasks with a v ent ca p (0.22 μm hydr ophobic membr ane).In vivo fluor escence (IVF) was measured (AquaFluor ®, Turner Designs) as a proxy for Synechococcus biomass.An aliquot of all culture treatments was inoculated in Luria-Bertani (LB, Miller) broth once per growth phase and incubated in the dark at 25 • C for 3 days to verify that the cultur es r emained axenic during eac h experiment.A cultur e was considered axenic if the absorbance, measured at 610 nm, did not incr ease significantl y ov er this time, whic h was the case for all samples.Over the growth curve, Synechococcus culture aliquots were collected, fixed (final concentration of 0.2% paraformaldehyde), and stored at −80 • C until cell abundance analysis using the Guava ® EasyCyte flo w c ytometer (Millipor e).Briefly, Synec hococcus was enumerated in unstained samples based on red fluorescence (i.e .c hlor ophyll) and forw ar d scatter signals using a lo w flo w rate of 0.24 μl s −1 for 1 min.Instrument-specific beads (Guav a ® Chec k Kit, Luminex) were used to calibrate the instrument.

Growth on DOP substrates
The capacity of Synechococcus WH8102 and WH5701 to grow on different DOP bond-classes as a sole P source was tested in −P i SN media amended with a single DOP substrate (45 μmol l −1 P, final concentration; Waterbury et al. 1986 ).To examine Synec hococcus gr owth acr oss DOP bond-classes , two P-monoesters , one P-diester, two P-anhydrides, and four phosphonates were selected as r epr esentativ es of the DOP pool.Additionally, a Pmonoester containing P-anhydride bonds was selected to examine growth on a DOP compound containing multiple bondclasses.Specificall y, r epr esentativ e DOP compounds included the P-monoesters glucose-6-phosphate (Glc-6-P) and adenosine 5monophosphate (AMP); the P-diester bis(4-methylumbelliferyl) phosphate (BisMUF-P); the short and long chain polyphosphates: 3-Pol yP and 45-pol yphosphate (45-Pol yP); the P-monoester and Panhydride containing adenosine 5 -triphosphate (ATP); and the phosphonates: 4-nitr ophen yl phen ylphosphonate (4-NpPn), 2aminometh ylphosphonic acid (2-AEPn), meth ylphosphonic acid (MPn), and ethylphosphonic acid (EPn).Two separate experiments were carried out in triplicate to test growth on (1) P-monoester and Pol yP substr ates, and (2) a P-diester and phosphonates.Two contr ol tr eatments (cultur e gr o wn in + P i and −P i media) w ere carried out in triplicate for each experiment.IVF was measur ed dail y in eac h tr eatment ov er ∼20 da ys , and cell axenicity was tested e v ery ∼5 da ys .Gr owth r ates wer e calculated as the slope of the best-fit line over the natural log-linear portion of the IVF curve (typically within days 0-7, except for AMP which was within days 7-13 to account for the delayed growth; Table S1 ).
To address the possibility of abiotic degradation of the amended DOP substrates under culture conditions, a separate experiment was conducted, measuring autohydr ol ysis of eac h DOP substr ate ov er 20 da ys .P i concentr ation was measur ed in −P i SN media amended with a single DOP compound (36 μmol l −1 ; final P concentration as in Diaz et al. 2018Diaz et al. , 2019 ) ) and no addition of cells ( Figure S1 ).Treatments were sampled immediately after DOP addition, and again every 5 days over a 20-day incubation period.Samples wer e fr ozen at −20 • C, and P i [or soluble r eactiv e phosphorus (SRP)] was measured using a standard colorimetric protocol (Hansen and Koroleff 1999 ) on a multimode plate reader Table 1.Compar ativ e anal ysis of Synechococcus culture strains' ATP and 3-PolyP hydrolysis rates.For each strain and media type, maxim um hydr ol ysis r ates for 3-Pol yP and ATP were selected (days 16-23 for WH8102 and days 7-17 for WH5701).The av er a ge r atio of ATP to 3-Pol yP hydr ol ysis (ATP:3-Pol yP) is expr essed as a percentage (%).P-anhydride bond degradation alone results in a hydr ol ysis percenta ge of 66.7%.A higher percentage indicates P-ester bond degradation from ATP. T scores (T; reported as absolute values) are calculated by subtracting the ATP:3-PolyP percentage from 66.7 and then dividing by the standard error (SE).The degrees of freedom (df) and two-tailed P-v alues ar e included.T he a v er a ge detection limit of P i using this method, defined as three times the standard deviation of the triplicate blank measurements, was 0.125 ± 0.005 μmol l −1 .The calibration curve was pr epar ed with 0.2 μm filtered ALOHA seawater with a P i backgr ound concentr ation below the detection limit.

DOP hydrolysis
The capacity of Synechococcus WH8102 and WH5701 to hydrolyze different DOP bond-classes was determined in the presence (whole cell) and absence (cell-free filtrate) of cells over the growth curve (Diaz et al. 2018(Diaz et al. , 2019 ) ).Some P-hydrolases are predicted to be ectoenzymes, and substantial cell-free P-hydrolase activity has been documented in diverse marine environments (Duhamel et al. 2010, Baltar et al. 2019 ).The inclusion of cell-fr ee filtr ate hydr ol ysis helped r e v eal the mec hanisms (enzymes) involv ed in DOP degradation.Since the selected strains did not grow on phosphonates, only DOP compounds with P-ester and P-anhydride bonds wer e tested.Thr ee substr ates wer e selected to conduct this experiment.The short-chain 3-PolyP was tested as the representativ e pol yphosphate substr ate (P-anhydrides onl y).Because nucleotides ATP and AMP shar e the same P-ester cor e and contain different bond types, they were also selected.AMP was selected as the r epr esentativ e P-ester, while ATP, containing both P-anhydride and P-ester bonds, served as a re presentati ve with both bondclasses.
Whole cell and cell-free filtrate experiments were carried out separ atel y.For both experiment types, Synechococcus strains were grown in triplicate + Pi and −Pi SN media.The + P i and −P i cultures were subsampled approximately every 3 days over ∼20 days to obtain whole cell and cell-free DOP hydrolysis results along each phase of the cellular growth curve.
To determine DOP hydr ol ysis r ates on eac h subsampling day, aliquots (200 μl) of + P i and −P i tr eatments wer e amended with a single DOP substrate (3-PolyP , ATP , or AMP; 20 μmol l −1 P, final concentration) in triplicate wells of a nontreated standard 96-well tr anspar ent micr oplate and incubated over 6-h.For whole cell subsamples, aliquots were directly taken from the culture flasks, while cell-free filtrate subsamples were prepared by aseptically filtering (0.2 μm) culture aliquots before DOP hydrolysis determination.The following contr ols wer e pr epar ed in triplicate wells of the microplate and monitored in parallel: (1) an unamended treatment (addition of cells and no DOP substrate) to track P i concentrations in the cultures over time, (2) a treatment amended with KH 2 PO 4 (45 μmol l −1 P, final concentration) to correct for the uptake/adsorption of P i released from DOP by the cells (Diaz et al. 2019 ), and (3) boiled (15 min) filtrates amended with DOP to assess potential autohydr ol ysis during the 6-h incubation of the plate.P i concentration was measured following the colorimetric protocol described abo ve .T he unamended treatment sho w ed negligible (below detection limit) P i release during the 6-h incubations for all subsamplings over the growth curve, ruling out the release of periplasmic (Kamennaya et al. 2020 ) and other cellular sources of P i as a major factor.DOP hydr ol ysis r ates wer e normalized to flow cytometry cell counts to account for biomass differences between strains and treatments.
To determine if the observed hydrolysis rates were sufficient to sustain cellular P demand (Diaz et al. 2019 ), the following equation was used: where H is the maximum hydrolysis rate (amol cell −1 day −1 ), Q is the P quota (amol cell −1 ), and g is the growth rate (day −1 ).P quota v alues wer e taken fr om the r ange of v alues r eported in Bertilsson et al. ( 2003 ), Heldal et al. ( 2003 ), Fu et al. ( 2006 ), and Lopez et al. ( 2016 ) for Synechococcus culture isolates (inclusive of WH8102, WH8103, WH8104, and WH7803; no published values are available for WH5701) grown in + P i and −P i conditions.Specifically, the minimum and maximum P quotas for cultures grown in + P i (58.0 and 140.0 amol cell −1 , r espectiv el y) and for cultures grown in −P i (16.0 amol cell −1 and 25.0 amol cell −1 , r espectiv el y) wer e used.For eac h observ ed r ate of hydr ol ysis, an expected gr owth r ate ( g ) was calculated using the maximum hydrolysis rates (days 11-21 for WH8102, days 11-20 for WH5701) and expressed as a proportion of the observed + P i growth rate.Ratios greater than 1 indicate that the observed DOP hydrolysis rates were sufficient to sustain P demand similar to + P i ( Table S2 ).
Compar ativ e anal ysis was carried out between ATP and 3-PolyP to determine if the P-ester bond of ATP is likel y degr aded alongside the P-anhydride bond (Table 1 ).Four consecutive subsampling da ys , encompassing peak hydr ol ysis r ates, wer e selected for eac h str ain and media type in the whole cell experiment.As ATP contains a P-ester and two P-anhydride bonds, the substrate provides bond-class hydr ol ysis on a single substrate.Under complete hydr ol ysis of either ATP or 3-Pol yP, thr ee orthophosphates can be released; ho w ever, a maximum of two and three orthophosphates from ATP and 3-polyP , respectively , can be released by hydr ol ysis of P-anhydride bonds alone .T her efor e, under complete P-anhydride degr adation, wher e the P-ester bond of ATP remains intact, the ATP:3-Pol yP hydr ol ysis r atio is 2:3 (or 66.7%) (Diaz et al. 2018 ).If the ATP:3-Pol yP hydr ol ysis r atio is > 66.7%, it suggests the likel y degr adation of the P-ester bond of ATP as well (Table 1 ).

Cell-free proteins
For Synechococcus extracellular protein identification following growth in minimal P, WH8102 and WH5701 were grown in triplicate 500 ml culture flasks in −P i SN media amended with 1 μmol l −1 KH 2 PO 4 ; a concentration previously determined as being low enough to induce P-stress while still maintaining biomass (Cox and Saito 2013 ).In the stationary phase, each culture triplicate was tr ansferr ed to an autoclav ed 250 ml pol ypr opylene bottle and centrifuged at 3200 r m −1 for 20 min at 4 • C. The supernatant was filtered using a sterile disposable v acuum filtr ation system (0.2 μm).Filtrate (70 ml) was added to a prerinsed Centricon spin column (Tris Buffer; 20 mmol l −1 , pH = 8) and centrifuged at 3200 r m −1 for 45 min at 4 • C. The pr ocedur e was r epeated thr ee times for both strains and triplicates .T he sample was then dialyzed twice with Tris buffer (20 mmol l −1 , pH = 8), and the concentrate was recov er ed and br ought to a final volume of ∼500 μl using Tris Buffer.A Quic kStart Br adford pr otein assay kit (Bio-Rad), calibrated using a standard curve of gamma globulin, was used to ensure that the samples contained enough protein (minimum of 100 μg ml −1 ) for further analyses.According to the manufacturer's instructions, a trypsin digest was carried out on 10.5 μl of each triplicate using an in-solution digestion kit (ThermoScientific).Peptide samples wer e anal yzed at the Pr oteomics and Mass Spectr ometry facility at the University of Georgia on a Thermo-Fisher LTQ Orbitrap Elite mass spectrometer coupled with a Proxeon Easy NanoLC system (Waltham, MA, USA) following Adams et al. ( 2022 ).Peptides were mapped to the Synechococcus genome (NCBI BioProject PRJNA230) (P alenik et al. 2003(P alenik et al. , McCarr en et al. 2005 ) ).The protein set was searched in BLASTP against NCBI nonredundant databases, and accession numbers were cross-referenced on UniProt to confirm putative function and identify APs.

MUF-P displacement
The affinity of Synechococcus APs for different DOP model substrates with varying P bond-classes was examined through its ability to inhibit the hydr ol ysis of the fluor ogenic substr ate 4methylumbelliferyl phosphate (MUF-P) (Nedoma et al. 2007 ).Lateexponential phase samples of −P i cultures were incubated in triplicate wells of a blac k, nontr eated 96-well microplate for each Synec hococcus str ain.Either a Pol yP (3-Pol yP, 45-Pol yP), P-ester (ATP, AMP, Glc-6-P), or phosphonate (MPn) was added in a series of concentrations (0, 2, 5, 10, 20, 40, 70, and 100 μmol l −1 ; final P concentration) with a single concentration of MUF-P.The selected DOP substrates exhibit no apparent chemical properties conducive to irr e v ersible binding (Reid and Wilson 1971, Holtz et al. 1999, Whisnant and Gilman 2002 ).The MUF-P concentration was 10% of the pr e viousl y determined Mic haelis-Menten constant ( K m ).This value was established by incubating 200 μl of each Synechococcus strain with a range of MUF-P concentrations (0 to 20 μmol l −1 ), following the equation: where S and V are the concentrations of MUF-P and the MUF-P hydr ol ysis r ates, r espectiv el y.
For each tested DOP model substrate, MUF-P hydrolysis velocity was determined fluor ometricall y (excitation/emission: 359/449, 4-methylumbelliferone -MUF), using a multimode plate reader (Spectr aMax ® M2, Molecular De vices) at m ultiple time points ov er an incubation period of 24-h to ensure linearity.MUF-P hydrolysis inhibition (%) is defined as a decrease of MUF-P hydr ol ysis v elocity ov er the tested r ange of model DOP substr ate concentr ations r elativ e to the contr ol without DOP (r eceiving onl y a single MUF-P concentration).To determine IC 50 , which corresponds to the DOP concentration that causes 50% inhibition of MUF-P, data (both MUF-P hydr ol ysis inhibition and DOP concentr ations) wer e fitted with a sigmoid function as described in Nedoma et al. ( 2007 ).Because the amended MUF-P concentration was 10% of the K m for both strains (i.e .10% of 5 μmol l −1 = 0.5 μmol l −1 ), the IC 50 value can be dir ectl y expr essed as the inhibition constant K i (Nedoma et al. 2007 ).As such, a DOP substrate with a low binding affinity will have a high K i , as a higher DOP concentration is required to inhibit MUF-P hydr ol ysis.

Data and statistical analyses
Statistical analyses for the displacement experiment were carried out in MATLAB.DOP concentrations that inhibit MUF-P hydr ol ysis velocities by 50% were compared using a one-way ANOVA.Flow c ytometry data w er e pr ocessed using FCS Expr ess 7.All r emaining data analyses were performed in Microsoft Excel and GraphPad Prism 8. Substr ates wer e compar ed following a tw o-w ay ANOVA and post hoc testing with Dunnet's method (between experimental and control treatments) to assess culture growth on DOP.Differences in cell-normalized hydr ol ysis r ates wer e e v aluated using r epeated measures ANOVA and post hoc testing with either Tuk e y's honest significant difference (between the two strains and substr ates under eac h gr o wth condition) or Dunnett's (betw een experimental and control treatments) method.

Growth on phosphoanhydrides, phosphoesters, and phosphonates
Axenic cultures of Synechococcus WH8102 and WH5701 grew on a v ariety of DOP compounds, inclusiv e of P-esters and P-anhydrides bond-classes, as a sole source of added P (Fig. 1 , Table S1 ).Both str ains gr e w on P-anhydride containing compounds 3-PolyP, 45-PolyP, and ATP with growth rates equivalent to ( P > .05;one-way ANOVA), or greater than ( P < .05;one-way ANOVA) the + P i treatment (i.e.> 0.5 day −1 ; Table S1 ).Additionall y, both str ains gr e w on the P-ester Glc-6-P similar to the + P i treatment ( P < .01 for WH5701, P = .3814for WH8102 ; one-way ANOVA).Howe v er, on the P-ester AMP, both str ains exhibited gr owth r ates significantl y lo w er than the + P i control ( P < .0001;one-way ANOVA).Specifically, for WH8102, growth on AMP was equivalent to growth on −P i ( P > .05;one-way ANOVA), and despite a slight increase in IVF for WH5701 on AMP by day 14, its growth r ate onl y r eac hed half that on + P i (Fig. 1 B).Neither str ain gr e w on phosphonates MPn, EPn, 2-AEPn, and 4-NpPn, with IVF v alues significantl y lo w er than the + P i control ( P < .05;tw o-w ay ANOVA, Fig. 1 C and D) and negligible gr owth r ates, similar to the −P i treatment ( < 0.15 day −1 ; Table S1 ).Growth on the P-diester BisMUF-P varied between str ains.For WH8102, IVF v alues steadil y incr eased, albeit lo w er than + P i and with a growth rate half that of + P i (0.20 ± 0.02 day −1 ; Table S1 ), while for WH5701, IVF values remained negligible over the growth curve and exhibited a growth rate (0.11 ± 0.01 day −1 ; Table S1 ) 3-fold lo w er than + P i , similar to −P i .Over the 20day autohydr ol ysis experiment, DOP with P-anhydride bonds (3-P olyP, 45-P olyP, and ATP) w ere autohydrolyzed.Lo w abiotic degradation occurred in the initial 5 days; by day 20, 19.0 ± 1.5 μmol l −1 of 3-PolyP, 9.0 ± 1.0 μmol l −1 of ATP, and 7.0 ± 6.0 μmol l −1 of 45-Pol yP wer e abioticall y degr aded ( Figur e S1 ).Ho w e v er, the rates of abiotic degradation were negligible compared to the whole cell DOP hydr ol ysis r ates measur ed ov er 6-h (see below).

DOP hydrolysis
To assess P-ester and P-anhydride degr adation, hydr ol ysis r ates of a r epr esentativ e P-anhydride (3-Pol yP) and P-ester (AMP), as well as a DOP compound with both P-anhydride and P-ester bonds (ATP), wer e measur ed for Synec hococcus WH8102 and WH5701 grown in + P i and −P i SN media (Fig. 2 ).All tested DOP substrates sho w ed autohydr ol ysis below the detection limit over the 6-h incubation for the whole cell and cell-free filtrate experiments.In the whole cell experiment, hydr ol ysis r ates per cell gener all y increased with time over the growth curve, consistent with the decrease in media P i concentration.Higher per-cell hydrolysis rates wer e observ ed in the −P i tr eatment r elativ e to + P i ( P < .05;oneway ANOVA; Fig. 2 ).Specifically, for WH8102, maximum hydrolysis rates for 3-PolyP , ATP , and AMP were ∼24-fold higher in the −P i treatment than in the + P i tr eatment.For WH5701, maxim um hydr ol ysis r ates in the −P i tr eatment wer e 32.0 ± 14.0-fold higher for 3-PolyP, 2.5 ± 1.0-fold higher for ATP, and 3.0 ± 2.0-fold higher for AMP, in comparison to the + P i treatment.Regardless of strain and P i av ailability, thr oughout the experiments , 3-P ol yP hydr ol ysis r ates wer e significantl y higher than A TP, and A TP hydr ol ysis r ates wer e significantl y higher than AMP ( P < .05;Fig. 2 C-F).Maximum DOP hydr ol ysis r ates wer e observ ed for 3-Pol yP in the −P i treatment at the onset of the stationary phase, r eac hing 502.0 ± 17.0 amol cell −1 h −1 for WH8102 and 406.0 ± 8.5 amol cell −1 h −1 for WH5701.
In the final 2 weeks, corresponding to the highest observed DOP hydr ol ysis r ates (Diaz et al. 2018 ), the ATP:3-Pol yP hydr ol ysis r atio was less than 2:3, or 66.7%, for both strains and P i treatments.This result suggests that the P-ester bond of ATP remained intact while the P-anhydride bond was hydr ol yzed (Table 1 ).In the early phase of the growth curve, AMP hydrolysis rates were below detection in both strains and P i treatments.By day 23 for WH8102 grown on + P i , low but consistent hydr ol ysis r ates wer e observ ed (4 ± 1 amol cell −1 h −1 ; Fig. 2 C).For the remaining strains and media types, while AMP hydr ol ysis was observ able on certain individual sampling da ys , r ates did not incr ease ov er time ( P > .05;tw o-w ay ANOVA; Fig. 2 D-F).
DOP hydr ol ysis activity was verified in the cell-free filtrate ( Figure S2 ), indicating the presence of cell-free P-hydrolase enzymes.DOP hydr ol ysis r ates in −P i filtr ates wer e observ ed ov er the growth curve ( P < .001;one-way ANOVA; Figure S2E and F ), with 3-Pol yP hydr ol ysis r ates significantl y higher than ATP hydr olysis r ates ( Figur e S2E and F ). AMP hydr ol ysis r ates wer e negligible in all filtr ates.Maxim um hydr ol ysis r ates observ ed in the filtr ates occurr ed for 3-Pol yP in the −P i tr eatment on day 21 (471.0 ± 11.0 amol cell −1 h −1 for WH8102, 220.0 ± 1.0 amol cell −1 h −1 for WH5701; Figure S2E and F ). DOP hydrolysis by WH8102 grown in + P i media was minimal over the growth curve, with no significant differ ence ov er time and between tr eatments ( P = .6250;Figur e S2C ).Hydr ol ysis r ates wer e minimal for WH5701 grown in + P i media, except on day 21 (64.0 ± 3.0 amol cell −1 h −1 for 3-PolyP, 23.0 ± 1.0 amol cell −1 h −1 for ATP, 1.0 ± 0.5 amol cell −1 h −1 for AMP; Figure S2D ).

Cell-free proteins
Ov er all, 43 and 25 pr oteins wer e pr esent in the exoproteome of P i -limited WH8102 and WH5701, r espectiv el y.Of that, 30% for WH8102 and 16% for WH5701 were hypothetical unidentified proteins ( Tables S3 and S4 ).Among the annotated proteins were ones associated with phosphate acquisition pathwa ys .A phosphate ABC transporter was identified in WH8102 (CAE07533.1)and WH5701 (EAQ75702.1).For WH8102, putative APs (CAE08906.DOP sources lacking a shared letter differ significantly ( P < .05).Days in which hydrolysis rates were not collected are denoted as "no data" (nd).

Discussion
The primary objective of this study was to assess the bond-specific bioavailability and utilization of DOP compounds to Synechococcus.
For both the open ocean strain (WH8102) and the coastal strain (WH5701), our results indicate a preference for the enzymatic hy- Figure 3. Synechococcus MUF-P hydrolysis inhibition by DOP.DOP substrates were applied at six different concentrations ( x -axis; 0-100 μmol l −1 ) in the presence of 4-methylumbelliferyl phosphate (0.5 μmol l −1 ; MUF-P) for P i -deplete Synechococcus WH8102 (A) and WH5701 (B).MUF-P hydrolysis, r epr esented as a percentage (%) of the control (no DOP addition), is displayed on the y -axis.Dots r epr esent the av er a ge of three biological replicates, and error bars are omitted for visual clarity.Biological triplicates typically agreed to within ± 9%.
Table 2. Inhibition constants for the displacement of MUF-P by DOP.Inhibition constants (K i ), which correspond to the concentrations necessary for half saturation of phosphatases by differ ent DOP substr ates, ar e pr esented for Synec hococcus str ains WH8102 and WH5701.DOP substrates included short and long chain polyphosphates: 3-P olyP and 45-P olyP; the P-monoester and P-anhydride containing adenosine 5 -triphosphate (ATP); the P-monoesters AMP and Glc-6-P; and phosphonate MPn.Treatments in which the DOP addition did not result in a 50% (at minimum) 4-methylumbelliferyl phosphate (MUF-P) inhibition are denoted as "not applicable" (n.a.).

Growth on phosphoanhydrides, phosphoesters, and phosphonates
Synechococcus is a widespread c y anobacterium that is fr equentl y exposed to low P i concentrations, suggesting the need for a mechanism to cope with P i scarcity (Lomas et al. 2010, Duhamel et al. 2014, 2021, Karl and Björkman 2015 ).DOP can serve as a P source in addition to P i , though the DOP pool contains a range of bond types of unknown bioavailability for the microbial community (Karl andBjörkman 2015 , Hull andRuttenberg 2022 ).Although it is widel y belie v ed that P-esters play a pr edominant r ole in micr obial interactions with DOP (i.e .Karl and Björkman 2015 ), our results indicate that Synechococcus consistently exhibits growth on DOP substrates containing P-anhydrides, shows negligible growth on phosphonates, and displays substrate-dependent growth on Pesters.
Pol yP onl y accounts for ∼8%-13% of high molecular weight dissolv ed or ganic matter (Young and Ingall 2010, Diaz et al. 2016, Saad et al. 2016 ), and 1%-5% of the DOP pool in coastal environments (Bell et al. 2017(Bell et al. , 2020 ) ).That said, both the open ocean and coastal Synechococcus strains exhibited robust growth when cultured with P-anhydride containing compounds (specifically, 3-P olyP, 45-P olyP, and ATP), comparable to their growth with P i as the sole P source (Fig. 1 ).These results indicate that the selected P-anhydride compounds are bioavailable to both str ains, whic h builds on prior results for WH8102 (Moore et al. 2005 ).While phosphonate utilization pathwa ys , suc h as tr ansport genes ( phnDCE ), have been previously identified for Synechococcus (Moore et al. 2005, Ilikchyan et al. 2009, Shah et al. 2023 ), neither strain grew on phosphonates as a sole P source.Similarly, Shah et al. ( 2023 ) recently identified a lack of WH8102 growth on methylphosphonate, pr ompting the r einter pr etation of its phnDCE genes as regulatory factors in P i transport.
Phosphoesters, including diesters and monoesters, comprise most of the high molecular weight dissolved organic matter ( ∼80%-85%; Young and Ingall 2010 ), as well as coastal environment DOP pool ( ∼60% and ∼30%, r espectiv el y; Bell et al. 2020 ).Phosphodiesterase and phosphomonoesterase activity in marine environments suggest that P-diesters are less bioavailable than Pmonoesters (Sato et al. 2013 ).Her e, Synec hococcus str ains exhibited v arying gr owth patterns when pr ovided the P-diester BisMUF-P.WH8102 displayed an initially stunted growth curve that eventuall y incr eased, r eac hing maxim um IVF v alues similar to P i .In contr ast, WH5701 demonstr ated minimal gr owth on BisMUF-P (Fig. 1 ) These r esults impl y that P-diesters may be more bioavailable to str ains fr om the open ocean, where P i is fr equentl y limited or colimited, compared to coastal environments (Moore et al. 2013 , Browning andMoore 2023 ).The delayed growth also suggests that for the open ocean strain, consistent and prolonged exposure to P-diesters may be r equir ed for the culture to employ a mechanism for utilization.This could include producing phosphodiesterases or AP that can hydr ol yze P-monoesters and P-diesters (Sriv astav a et al. 2021 ).
Synec hococcus str ains displayed div erse gr owth r esponses when exposed to P-monoesters.Both strains grew on Glc-6-P similar to the + P i treatment ( Table S1 ); a result that aligns with Synechococcus WH7803 P i cleav a ge of Glc-6-P (Donald et al. 1997 ).Ho w e v er, WH8102 failed to grow on AMP, and although WH5701 sho w ed slight growth on AMP, it was delayed, and far less than the + P i treatment (Fig. 1 ).This is in agreement with the low and undetectable rates of AMP hydrolysis in cultures grown in + P i or −P i conditions (Fig. 2 ).The delay ed gro wth on AMP implies that similar to P-diester BisMUF-P, the culture may require prolonged and consistent exposure to AMP for sustained growth.It is possible that WH5701 could utilize AMP via direct uptake and/or low (undetectable) rates of enzymatic hydrolysis.Ho w ever, for E. coli , enzymatic cleav a ge of AMP by 5 -nucleotidase (5 -NT) is necessary for subsequent uptake of P i (Yagil and Beacham 1975 ), and to our knowledge, there is no evidence for AMP direct uptake by Synechococcus .Although 5 -nucleotidase is present in the Synechococcus genome, it was not identified in either strain in the exoproteome anal ysis.Ov er all, these r esults suggest that differ ent P-esters exhibit varying levels of bioavailability, emphasizing the importance of substrate diversity and complexity of P cycling in marine environments.

DOP hydrolysis fulfilling P demand
In a gr eement with their gr owth on P-anhydride substr ates as a sole P source, both −P i strains hydrolyzed 3-PolyP and ATP.Hydrolysis rates of 3-PolyP and ATP consistently and largely exceeded P demand for growth, in both strains grown in + P i or −P i media, and assuming a range of P quotas ( Table S2 ).For WH8102 in + P i , AMP hydr ol ysis was sufficient in meeting the P demand only if the minimum P quota was assumed, while for WH5701 in + P i , hydr ol ysis r ates wer e insufficient r egardless of the P quota.AMP hydr ol ysis was not detectable for WH8102 and WH5701 in −P i until day 21 and 20, r espectiv el y, at whic h point, despite being substantiall y lo w er than 3-PolyP and ATP hydr ol ysis, met the P demand.Cultur e gr owth on DOP substr ates was onl y monitor ed for 14 da ys , at which point cultures on + P i and DOP, supporting equal growth, had r eac hed the stationary phase for se v er al da ys .T her efor e, it is possible that longer incubations would have been necessary to observe sustained growth of both strains on AMP.DOP hydr ol ysis was observed the growth curve even in the + P i media with bac kgr ound P i concentrations at 34.0 ± 1.0 μmol l −1 for WH5701 and 29.0 ± 1.5 μmol l −1 for WH8102 by day 14; Fig. 2 A and B).Pr e vious studies documented DOP hydr ol ysis in natural communities under relatively high P i concentrations (Benitez-Nelson andBuesseler 1999 , Nausch et al. 2018 ).The measurable release of P i into the media indicates a lack of strict coupling between DOP hydr ol ysis and P i uptake .T hese combined results suggest that Synechococcus P-hydrolase enzymes, whether cell-free or cell-associated, liberate P i into their environment, potentially offering a source for assimilation by other microbes .T hese results are consistent with the high AP activity measur ed acr oss div erse natur al envir onments with differ ent P i concentrations (Duhamel et al. 2011, Thomson et al. 2019 ).Given that gr owth r ates on the thr ee P-anhydride substr ates either equaled or surpassed those on the + P i control and considering that the hydr ol ysis of DOP containing P-anhydride bonds exceeded the estimated P growth demand, these combined results strongly support the capability of both strains to thrive on P-anhydride compounds.

Preferential hydrolysis of the phosphoanhydride bond
Acr oss str ains and P i av ailability, 3-Pol yP hydr ol ysis r ates wer e higher than ATP hydr ol ysis r ates, and both wer e higher than AMP hydr ol ysis r ates, confirming the pr efer ential hydr ol ysis of the P-anhydride bond.Compar ativ e anal ysis between ATP and 3-PolyP aims to assess whether the P-ester bond of ATP degrades alongside the P-anhydride bond (Diaz et al. 2018 ).For each strain and media type, the ATP:3-Pol yP hydr ol ysis r atio consistentl y r emained below 2:3.This observation suggests that the P-anhydride bond of ATP was degraded while the P-monoester bond of ATP remained intact.Similar results have been reported in the case of the open ocean diatom Thalassiosira sp.CCMP1005 and CCMP1014, as well as coastal Thalassiosira sp.CCMP 1335 (Diaz et al. 2018 ).These consistent patterns underscore the potential significance of the P-anhydride bond specificity in microbial DOP bioavailability.

Cell-free filtr a te P-Hydrolases
Marine plankton can obtain P from the hydrolysis of DOP using their cell-surface-associated enzymes (Cembella et al. 1984 , Ammerman andAzam 1985 ).Some of these enzymes can also be liber ated fr om the cell by secr etion or upon death (cell l ysis or sloppy feeding), which contributes to the cell-free P-hydrolase activity measur ed acr oss P i -r e plete and P i -de plete marine environments (Duhamel et al. 2010, Baltar et al. 2019 ).Using cell-fr ee cultur e filtr ates fr om both str ains of Synec hococcus , w e found negligible hydr ol ysis of the three tested DOP substrates under + P i treatment but high hydr ol ysis r ates of 3-Pol yP and ATP in the −P i cultures ( Figure S2 ), confirming the presence of enzymes that act on DOP.While the cell-free filtrate is expected to include a mixture of natur all y and artificiall y r eleased extr acellular enzymes (i.e. via filtration), this method allows us to narrow down the enzymes potentiall y involv ed in DOP hydr ol ysis.
Both Synechococcus strains hydrolyzed PolyP in the whole cell and cell-free experiments, suggesting the presence of an enzyme that can specifically hydrolyze P-anhydrides .T he exoproteome of P i -depleted strains revealed AP isoforms phoX and phoA (for WH8102) and unidentified AP (for WH5701; accession EAQ75607.1).The pr e viousl y r eported genome of both strains contain enzymes known for acting on PolyP and P-esters, including AP isoforms phoA and phoX; and 5 -nucleotidase (5 -NT) and pol yphosphatase (ppX) (Moor e et al. 2005, Scanlan et al. 2009, Tetu et al. 2009, Kuto va ya et al. 2013, Christie-Oleza et al. 2015 ).Our results did not have PolyP-specific enzymes, so the observed extracellular Pol yP hydr ol ysis is likel y not dominated by Pol yP-specific enzymes .T his result aligns with the outcome of Adams et al. ( 2022 ).It does not exclude the possibility of direct low molecular weight PolyP uptake followed by interactions with ppK (synthesis and degradation) (Parnell et al. 2018 ) and ppX (degradation) or the potential for a low biomass sample (r esulting fr om gr owth on minimal P i ) to impact the detection of additional enzymes in our samples.

AP flexibility
Following the presence of APs in both Synechococcus strains, matc hed with 3-Pol yP and ATP hydr ol ysis and the absence of PolyP specific enzymes, it is likely that APs play a role in the hydr ol ysis of P-esters and PolyP in Synechococcus .Though it is generally assumed that APs onl y hydr ol yze P-monoesters (Tiwari et al. 2015 ), there is increasing evidence of AP substrate flexibility.APs from E. coli and calf intestine can cleave P-anhydride bonds (Yoza et al. 1997, Huang et al. 2018 ).Long-c hain Pol yP substr ates, up to 800 monomers , ma y be clea ved by mammalian AP depending on the concentration and ambient pH levels, indicating that AP substrate specificity may vary (Lorenz and Schröder 2001 ).To test this, we determined the ability of P-ester, P-anhydride, and phosphonate substrates to compete with MUF-P for the reaction of APs from Synechoccocus (Fig. 3 , Table 2 ).
The MUF-P displacement experiments provide insight into the r elativ e affinity of AP for the DOP bond-classes.All tested substr ates inhibited MUF-P hydr ol ysis by WH8102, indicating that Synechococcus WH8102 APs have broad substrate specificities (Fig. 3 , Table 2 ), similar to r esults r eported fr om the bacterial copiotroph R. pomeroyi (Adams et al. 2022 ).Specifically, P-esters Glc-6-P and AMP exhibited the highest MUF-P inhibition, follo w ed b y ATP and short-chain 3-P olyP.Long-chain 45-P olyP and MPn also resulted in a decrease in MUF-P hydrolysis.Ho w ever, 100 μmol l −1 of each substrate was required to reach the result of other Panhydrides at 30 μmol l −1 , suggesting a low AP-binding affinity for both long-chain polyphosphate and phosphonate MPn.These results suggest a short-chain PolyP preference by Synechococcus WH8102 APs .T his contr adicts the r esults of R. pomeroyi (Adams et al. 2022 ), possibly indicating different AP flexibilities across micr obial gr oups.
For WH5701, the response to DOP was muted, suggesting a lo w er AP affinity for the selected DOP substrates in the tested concentr ation r ange.At high DOP concentr ations (100 μmol l −1 ), P-ester and Pol yP substr ates decr eased MUF-P hydr ol ysis, though less than 50%, while phosphonate MPn did not.Since we measured high hydrolysis rates of ATP and 3-polyP via P i production assa ys , the o v er all low AP affinity for these substrates suggests that WH5701 likel y pr oduces enzymes that can hydr ol yze Pol yP and P-esters better than synthetic MUF-P.This result also highlights the importance of studying m ultiple str ains within Synechococcus when considering enzymatic responses.

Conclusion
There is growing evidence that phosphoanhydrides, in particular P olyP, pla y a pivotal role in the bioavailable pool of marine P (Martin et al. 2014, Diaz et al. 2018, 2019, Li et al. 2019, Sanz-Luque et al. 2020 ).Pol yP emer ges as a crucial nutritional P source, particularly under P i deficiency, supporting the growth of eukaryotic phytoplankton (Diaz et al. 2018(Diaz et al. , 2019 ) ), heter otr ophic bacteria (Adams et al. 2022 ), and Synechococcus WH8102 (Moore et al. 2005 ).This study further elucidates the importance of PolyP in open-ocean and coastal strains of Synechococcus and characterizes the potential for AP to drive its bioavailability and utilization.Our findings ec ho pr e vious observ ations of the pr efer ential degr adation of Panhydrides over P-esters, a phenomenon observed in diatoms of the genus Thalassiosira (Diaz et al. 2018(Diaz et al. , 2019 ) ), thus suggesting that P-anhydrides may substantially contribute to the nutritional DOP demand of phytoplankton.The r elativ el y low concentr ation of Panhydride containing compounds in natural marine DOP standing stocks could be explained by rapid microbial cycling (Young and Ingall 2010, Martin et al. 2014, Diaz et al. 2016, Bell et al. 2017, 2020 ).While AP activity has tr aditionall y been a measure of Pester hydr ol ysis, our study underscor es its substantial substr ate flexibility, including for short-chain P-anh ydride h ydr ol ysis.Notabl y, this flexibility v aries between the Synec hococcus WH8102 and WH5701 str ains.A compr ehensiv e understanding of DOP bioav ailability necessitates the c har acterization of the enzymes involved.This study emphasizes the importance of further c har acterizing these enzymes, including AP flexibility for different DOP bondclasses and within taxonomic groups .T hese insights contribute to our understanding of marine nutrient cycling and have implications for ecosystem dynamics and biogeochemical processes.

Figure 1 .
Figure1.Synec hococcus gr owth on DOP.Synechococcus WH8102 (A) and (C) and WH5701 (B) and (D) were grown on a single DOP substrate as the sole P source in two experiments, assessing IVF (RFU; y -axis) over time (day; x -axis) for P-monoesters and polyphosphates (A) and (B), as well as a P-diester and phosphonates (C) and (D).Re presentati ve DOP compounds included the P-monoesters Glc-6-P and AMP; the P-diester BisMUF-P; the short and long chain polyphosphates: 3-PolyP and 45-PolyP; the P-monoester and P-anhydride containing ATP; and the phosphonates: 4-NpPn, 2-AEPn, MPn, and EPn.Error bars indicate one standard deviation of the mean of three biological replicates.All symbols not visible at the x -axis sho w ed negligible gr owth, not significantl y differ ent ( P > .05)fr om the −P i tr eatment.

Figure 2 .
Figure2.Synechococcus DOP hydrolysis.SRP concentrations ( μmol l −1 ; squares) and cell abundance ( ×10 8 cells ml −1 ; circles) in P i -replete ( + P i ; filled symbols) and P i -deplete ( −P i ; empty symbols) media are displayed over time (day) for WH8102 (A) and WH5701 (B).P-hydr ol ysis r ates on selected model DOP substrates (3-PolyP , ATP , and AMP; amol cell −1 h −1 ) in + P i (C) and (D) and −P i (E) and (F) are normalized to cell abundance.P-hydrolysis rates ar e r epr esented as ov erla pping bars.Err or bars indicate one standard de viation of the mean of biological triplicates.Statistical r esults fr om r epeated measures ANOVA are provided above each P hydrolysis plot (C)-(F), indicating the significance of DOP hydrolysis rates throughout the experiment.Results from the pairwise post hoc comparison of each DOP source via Tuk e y's honest significant difference test are provided next to the legend entries.DOP sources lacking a shared letter differ significantly ( P < .05).Days in which hydrolysis rates were not collected are denoted as "no data" (nd).